Treating male infertility secondary to sperm oxidative stress

ABSTRACT

A profertility composition for administration to infertile men with sperm oxidative stress is disclosed. The composition comprises a unique combination of a pharmaceutically acceptable vitamin E, vitamin C, selenium, zinc, folic acid, lycopene and at least one carnitine source.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/174,732 filed May 1, 2009, the contents of which is herebyincorporated by reference in its entirety.

FIELD OF INVENTION

The present invention relates to methods, compositions and fertilitykits used to enhance the natural fertility potential of men. The presentinvention utilizes naturally occurring antioxidants as to provide aprophylactic treatment of sperm oxidative stress. Additionally, thenovel products described herein further include vitamins and mineralsfor improving semen parameters enhancing the overall health of spermthus increasing, fertilization rates, number of viable embryos,pregnancy rates and reducing miscarriages.

BACKGROUND

Male infertility has been on the rise for several decades and is nowconsidered a major health problem affecting around one in twelve of themen in USA. Id. If the trends observed over the last century continues,some infertility experts predict that by the middle of this century,most men will be advised to freeze their sperm at a young age to ensureadequate sperm quality.

A successful fertilization and subsequent embryo development requiresproperly matured sperm chromatin with minimal damage to its DNAintegrity. Equally important, is the integrity of sperm membrane lipidstructure essential for sperm motility and sperm-oocyte fusion. At leastone of the causes of sperm DNA damage and membrane peroxidation isattributed to excess Reactive Molecular Species (“RMS”), mostimportantly Reactive Oxygen Species (“ROS”), leading to a conditioncommonly referred to as sperm oxidative stress. In general, the term“Oxidative Stress” refers to a condition that occurs as a result ofexcessive generation of ROS and/or a diminished capacity of ROSscavenging endogenous antioxidants. Accordingly, the excessconcentration of ROS overwhelms the local environmental antioxidantdefense mechanism of the spermatozoa potentially leading to theformation of sperm cells with lower or no fertilizing potential.

Under normal conditions, cells utilize an array of enzymatic andnon-enzymatic antioxidants as internal defense to scavenge andneutralize excess levels of ROS. It is believed that oxidative stressdevelops either because of an overabundance of ROS from environmental orpathological stressors or a lack of antioxidant capacity to neutralizeexcess concentrations of ROS. Excess ROS within semen and the sperm cellhas been described to cause degradation of the cellular components suchas membrane and DNA, hence, the sperms injured by such oxidative specieshave diminished or no fertility potential.

To date several studies have reported that levels of ROS within semencan be reduced by augmenting the scavenging capacity of seminal plasmausing oral antioxidant supplementation. In fact, the benefits of oralantioxidant therapy has been described in numerous articles. By the wayof reference, such articles include Comhaire et al, The Effects ofCombined Conventional Treatment, Oral Antioxidants and Essential FattyAcids on Sperm Biology in Subfertile Men, Prostaglandins Leuko EssentFatty Acids, 63, 159-165 (2000); Lenzi et al, Placebo-Controlled,Double-blind, Cross-over Trial of Gluthathione therapy in maleinfertility, Hum Reprod, 8, 1657-1662 (1993); Greco et al, Reduction ofthe Incidence of Sperm DNA Fragmentation by Oral Antioxidant Treatment.J Androl, 17, 276-287 (2005); Tremellen et al, A Randomised ControlTrial Examining the Effect of an Antioxidant (Menevit) on PregnancyOutcome During IVF-ICSI Treatment, Australian and New Zealand Journal ofObstetrics and Gynecology 47, 216-221 (2007); and Agarwal et al,Oxidative stress, DNA Damage and Apoptosis in Male Infertility, aClinical Approach, BJU Int, 95, 503-507 (2005), the teachings of whichare incorporated herein in their entirety.

However, there is no product in the market today that has the instantlydisclosed combination of antioxidants, minerals and vitamins. Moreover,the prior art does not teach the steps described herein to enhance thenatural fertility process. At least one object of the present inventionis to address such a need in the field.

Excessive concentration of ROS can cause extensive sperm DNA damage andmembrane peroxidation. In general ROS includes reactive moieties such ashydroxyl radicals (OH⁻), ionic species such as superoxide anions (O₂ ⁻),and neutral but highly reactive oxidizing molecules such hydrogenperoxides (H₂O₂). In semen, such species are predominantly generated byleukocytes or morphologically abnormal sperms. It has been described inthe art that ROS plays both physiological and pathological roles inmaturation of sperm cells. For example, at normal baseline ROSconcentrations, sperms are properly matured as ROS modulates theregulation of the normal sperm function in various cascades includingbut not limited to sperm-oocyte fusion, acrosome reaction and spermcapacitation.

On the other hand, excess ROS concentrations result in direct insult oncellular components such as lipids, proteins and DNA, thus interferingwith normal cell function. Traditional semen analysis methodologies failto detect DNA damage caused by ROS. In fact, in many cases infertile menwho undergo a clinical workup show normal sperm parameters, includingsperm count motility and morphology. However, when compared with healthyfertile men, at least a subpopulation of these infertile men, showhigher concentrations of oxidants in their seminal fluid which may becoupled with significantly lower concentrations of antioxidant ascompared to healthy fertile men.

At the genomic level, excess concentrations of ROS modifies sperm DNAstructure in a number of ways. For example, ROS can induce chromatincross-linking, DNA base oxidation and cause high frequencies of singleand double DNA strand breaks. Even though, sperms with damaged DNA maystill fertilize the oocyte, the degree of DNA damage can have profoundimplications in normality of embryonic development resulting in highermiscarriage rates, birth defects or the long-term health of the progeny.

Current medical approach to facilitate embryonic development andpregnancy uses invasive techniques collectively referred to as AssistedReproductive Techniques (“ART”), including Intrauterine Insemination(“IUI”), In vitro Fertilization (“IVF”) and Intracytoplasmic SpermInjection (“ICSI”). However, such techniques generally have an averagerate of success of about 30%. At least one explanation for such pooroutcome is the fact that the semen and the sperm analysis techniquesemployed during ART cannot differentiate sperms with DNA damage from thenormal ones.

Moreover, ART modes of fertilization have been linked with birth defectsand incidences of childhood cancer, e.x. acute leukemia and lymphoma.Nevertheless, ART remains the only viable option for infertile couples.Another shortcoming of the ART is the cost. Couples on average undergoseveral IVF attempts to achieve a pregnancy, therefore undertaking hugeexpenses. Additionally, the risk of failure often causes intenseemotional trauma.

Moreover, ART can not and does not address the damage done by ROS tosperm and ultimately to the fertilized egg. Therefore, there is a needin the art to address the impact of Sperm Oxidative Stress (“SOS”) amonginfertile couples. Currently, there are no effective and generallyaccepted pharmacotherapy that can combat the problem of male infertilityassociated with SOS. Thus, there is a need for convenient, inexpensivefertility methodologies and products to aid and improve couple'sfertility potential. There is also a need for a kit that will providecouples in need with all products, tools and material required tofacilitate a comprehensive plan for achieving a successful pregnancy.

SUMMARY OF THE PRESENT INVENTION

The present invention fills the foregoing need by lowering andprophylactically treating SOS by boosting the non-enzymatic antioxidantdefense mechanism. The invention comprises unique combinations ofseveral natural antioxidants, vitamins and minerals clinically tried forefficacy to improve sperm health and quality. The invention is thereforeintended to be a prophylactic treatment to improve the overall health ofsperms thus improving the sperm-oocyte fertilization, reducingmiscarriage rates and improving pregnancy rates.

This invention provides methods of using combinations of naturallyoccurring antioxidant molecules for treating infertility in menpredominantly suffering from SOS alone or in combination with aromataseinhibitors, suitable anti-inflammatory, and/or antibiotics.

At least one aspect of the present invention is directed to a method ofincreasing the rate of pregnancy in a female subject by administering tothe male partner an effective amount of a combination of anti-oxidants,vitamins and minerals, for at least a period of up to about three (3)months prior to any fertilization attempts, wherein said combination issubstantially free of vitamin A, vitamin K and/or garlic. In at leastone embodiment of this aspect of the invention, the combination includesat least one source of vitamin C, at least one source of vitamin E, atleast one source of carnitine, lycopene and at least one source of folicacid.

In a more preferred embodiment of this aspect of the invention, thecombination includes at least one source of vitamin C, at least onesource of vitamin E, at least one source of carnitine, and at least onesource of folic acid, at least one mineral which is selenium, zinc,magnesium or combinations thereof, wherein the composition is free ofany traces of vitamin A, vitamin K and/or garlic.

In another aspect of the present invention, a novel method of reducingROS damage to sperm DNA by at least 2.5% as compared with placebo,including the steps of administering to a male subject for at least aperiod of up to about three (3) months an effective amount of thecombination of anti-oxidants, vitamins and minerals disclosed herein,wherein the combination is free of vitamin A, vitamin K and/or garlic.

In at least one embodiment of this aspect of the invention, the methodof inhibiting ROS damage to sperm DNA cause a reduction in a measurementof DNA fragmentation secondary to SOS by at least 1%, 2.5%, 5%, 8%, 10%,12.5%, 15%, 20%, 30% and preferably higher when compared to a placebotreatment. In another embodiment, the sperm DNA fragmentation isassessed by such technique including but not limited to Sperm ChromatinDispersion (“SCD”) test, Sperm Chromatin Structure Assay (“SCSA”), DNABreakage Detection-Fluorescentce In Situ Hybridization (“DBD-FISH”)test, Terminal Deoxyribonucleotidyl Transferase-Mediated dUTP Nic-EndLabelling (“TUNEL”) assay and 8-hydroxy-deoxyguanosine Assay (“8-OH-dGAssay”).

Another aspect of the present invention provides for a method oftreating an infertile male subject with sperm oxidative stress includingthe steps of (a) ascertaining the degree of DNA damage in said malesubject, as measured by SCSA, TUNEL, SCD, DBD-FISH, or 8-OH-dG Assay,(b) initiating a course of therapy by administering an effective amountof a combination of anti-oxidants, vitamins and minerals and free ofvitamin A, vitamin K and/or garlic for at least a period of three (3)months, (c) reascertaining the degree of DNA damage in said male subjectpost the therapy of the step (b), and (d) fertilizing the egg of thefemale partner with said male subject's sperm after completion of thetreatment course of step (b), wherein the degree of the DNA damagemeasurement is reduced or otherwise improved, as compared to placebo orsaid subject's own pretreatment values by at least 1%, 2.5%, 5%, 8%,10%, 12.5%, 15%, 20%, 30%, and preferably higher.

Another aspect of the present invention provides for a method oftreating an infertile couple including the steps of (a) determining thecouples susceptibility to oxidative stress, (b) initiating thecombination regimen disclosed herein for the male partner wherein themale partner receives an effective amount of a combination ofanti-oxidants, vitamins and minerals and free of vitamin A, vitamin Kand/or garlic for at least a period of three (3) months. In anotheraspect of the present invention, the female partner may independently orsimultaneously with the male partner receive the anti-oxidant therapyoptionally including an alpha-lipoic acid regimen.

The present invention also provides a method of reducing theconcentration of ROS generated by leukocytes in the reproductive tractand/or semen of a male subject, the method including the steps ofadministering to the male subject (a) an effective amount of theantioxidant, vitamin and mineral combination for at least a period ofthree (3) months and (b) an effective amount of an aromatase inhibitoragent or an appropriate antibiotic.

The present invention also provides a method of improving sperm functionin a male subject, the method including the steps of administering tothe male subject an effective amount of the combination described hereinfor at least a period of three (3) months.

The present invention also provides for a method of improving quality ofan embryo produced by fertilization of an oocyte by a sperm from a malesubject, wherein the male subject has completed a three (3) month courseof antioxidant regimen comprising receiving a combination of at leastone source of vitamin C, at least one source of vitamin E, at least onesource of carnitine, and at least one source of folic acid, at least onemineral which is selenium, zinc, magnesium or combinations thereof,wherein the regimen is free of any traces of vitamin A, vitamin K and/orgarlic.

In at least one embodiment of this aspect of the invention, methods ofprophylactically reducing the risk of forming a nonviable zygotecomprising administering to a male subject in need thereof, acomposition comprising an effective amount of a combination of at leastone source of vitamin C, at least one source of vitamin E, at least onesource of carnitine, and at least one source of folic acid, at least onemineral which is selenium, zinc, magnesium or combinations thereof,wherein the regimen is free of any traces of vitamin A, vitamin K and/orgarlic, and optionally an effective dose of an aromatase inhibitor or anantibiotic.

The present invention also provides a composition comprising aneffective amount of at least one source of vitamin C, an effectiveamount of at least one source of vitamin E, an effective amount of atleast two sources of carnitine, and an effective amount of at least onesource of folic acid, at least one mineral which is selenium, zinc,magnesium or combinations thereof wherein the composition issubstantially free of any traces of vitamin A, vitamin K and/or garlic.In a preferred embodiment of this aspect of the invention, thecomposition only contains an effective amount of at least a source ofvitamin C, an effective amount of at least one source of vitamin E, aneffective amount of L-carnitine, propionyl-L-carnitine oracetyl-L-carnitine or combinations thereof, and an effective amount ofat least one source of folic acid, an effective amount of lycopene, andan effective amount of at least one mineral which is selenium, zinc,magnesium or combinations thereof, wherein the composition is free ofany traces of vitamin A, vitamin K and/or garlic.

In a preferred embodiment of this aspect of the present invention,inventor discloses a composition that contains vitamin E, vitamin C or asuitable salt thereof, folic acid, lycopene, a carnitine source,selenium, and zinc in pharmaceutically acceptable forms wherein theformulation is substantially free of vitamin A and vitamin K and/orgarlic or any extracts thereof.

In a more preferred embodiment of this aspect of the invention thecomposition is composed of vitamin E in about 100-800 IU; vitamin Cabout 50-800 mg, or an equivalent salt thereof; selenium in about 25-100μg; zinc in about 10-35 mg; folic acid in about 0.25-0.75 mg; lycopeneabout 3-12 mg; and a carnitine source in about 250-4000 mg, wherein theformulation is substantially free of vitamin A and vitamin K and/orgarlic or any extracts thereof. In yet another embodiment of the presentinvention, the carnitine source is a prodrug for carnitine.

In the preferred embodiment of the present invention the compositionincludes vitamin E in about 200 IU; vitamin C in about 90 or 200 mg,selenium in about 55 μg; zinc in about 15 mg; folic acid in about 0.5mg; lycopene about 5 mg; and acetyl-L-carnitine in about 1000 mg. In yetanother embodiment, the composition includes vitamin E in about 200 IU;vitamin C in about 200 mg, selenium in about 55 μg; zinc in about 15 mg;folic acid in about 0.5 mg; lycopene about 5 mg; andpropionyl-L-carnitine in about 500 mg, and acetyl-L-carnitine in about1000 mg.

Another aspect of the present invention is directed to the disclosedantioxidant containing compositions, wherein the compositions are in theform of a tablet, a capsule, a powder mixture, a paste, a suspension, asolution, an elixir, a topical or an oral patch, or a parenteralpreparation. In at least on embodiment of this aspect of the invention,the composition contains sustain or extended release components,immediate release components, microparticles, nanoparticles and suitableexcipients to enhance the bioavailability of the product as compared totheir respective conventional preparations. In the preferred embodimentof this aspect of the invention, the antioxidant containing compositioncan be administered orally, via an inhaler, or via the parenteral route.

In another aspect of the present invention, pregnancy kits are providedcomprising a set of pregnancy tests, articles for storage of specimens,and compositions containing vitamin E in about 200 IU; vitamin C inabout 90 or 200 mg, selenium in about 55 μg; zinc in about 15 mg; folicacid in about 0.5 mg; lycopene about 5 mg; and acetyl L-carnitine inabout 500 mg and/or compositions containing vitamin E in about 200 IU;vitamin C in about 90 or 200 mg, selenium in about 55 μg; zinc in about15 mg; folic acid in about 0.5 mg; lycopene about 5 mg; andpropionyl-L-carnitine in about 500 mg.

In at least another aspect of the present invention, pregnancy kits cancomprise additional adjuvant agents such as aromatase inhibitors,glutathiones, or Coenzyme Q10, each of which may be administeredseparately or in combination with the antioxidant, mineral and vitamincombinations instantly disclosed.

The present invention also provides a method of isolating sperm and/ormeasuring DNA fragmentation from a male subject, the method includingthe steps of (a) administering to the male subject an effective amountof at least one source of vitamin C, an effective amount of at least onesource of vitamin E, an effective amount of at least one source ofcarnitine, and an effective amount of at least one source of folic acid,an effective amount of lycopene, at least one mineral which is selenium,zinc, magnesium or combinations thereof wherein the composition issubstantially free of any traces of vitamin A, vitamin K and/or garlicand (b) measuring the subject's sperm's DNA fragmentation index.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs and shall be understood to have themeanings described below. All publications and patents referred toherein are incorporated by reference in their entirety. Unless otherwisespecified, a reference to a particular compound includes all ionic,salt, solvate (e.g., hydrate), protected forms, prodrugs, and otherstereoisomers thereof, isomeric forms, including racemic and othermixtures thereof. The invention provides the first products to put thesecomponents together to act in a synergistic manner in men'sformulations. The invention also provides for the very first time theunexpected reduction of the DNA sperm fragmentation by the combinationdescribed herein:

The term “aromatase inhibitors” is used herein to refer to reversible orirreversible aromatase inhibitors including but not limited to naturalflavanoids or synthetic products such products as anastrozole andexemestane (which is sold as Aromasin and is chemically described as6-methylenandrosta-1,4-diene-3,17-dione), analogues or derivativesthereof such as those described in U.S. Pat. Nos. 4,808,616, 4,876,045and 4,904,650, the teachings of which are incorporated herein in theirentirety.

As used herein, “about” will mean up to plus or minus 5% of theparticular term.

As used herein, “consisting essentially of” refers to excluding otheractive ingredients but including excipients.

The term “carnitine,” except as individually recited, includes allpharmaceutically acceptable forms of carnitine including but not limitedto 1-carnintine or a prodrug thereof, acetyl-L-carnitine andpropionyl-L-carnitine in form of HCl salt, in general alkanoylL-carnitines such as isovaleryl-L-carnitine, hexanoly-L-carnitine,octanoyl-L-carnitine, myristoyl-L-carnitine, palmitoyl-L-carnitine,stearoyl-L-carnitine.

By “Fertilization Attempt” it is meant any process that facilitatesfusion of the sperm and egg. Such term includes natural as well asmedical procedures that facilitate embryonic development and pregnancyincluding but not limited to IUI, IVF and ICSI.

The term “folic acid or folate” (sometimes known as vitamin B9) is meantto include the synthetic form of the vitamin, in hydrate or salt formswith metal ions such as sodium, zinc etc.

The term “lycopene” is meant the red pigment in tomatoes in itspharmaceutically acceptable organic or inorganic form, which contains aC₄₀ open-chain hydrocarbon carotenoid with any of its 11 conjugateddouble bonds being available for the formation of cis andtrans-geometrical isomers.

By the term “Reactive Molecular Species (“RMS”) it is meant to includeReactive Oxygen Species (“ROS”), Reactive Nitrogen Species (“RNS”) andother reactive heteroatom species that can incapacitate sperm ability tofertilize an egg, preferably ROS as the most evaluated reactivemoieties.

The term “selenium” is meant to include elemental, all inorganic andorganic forms of selenium such as sodium selenite and sodium selenate,selenomethionine, selenocysteine, methyselenocysteine, methylselenol,amino acid chelates, yeast, and kelp bound selenium.

By the term “substantially free” it is meant that the final product isfree of any traces of the identified ingredient to the extend that itcould be detected by general quantitative assays known in the art fordetection of such ingredient in the final product or if detected, it isin residual content of the ingredient and in principle only attributableto impurities.

The term “treatment” or “therapy” as used herein in the context oftreating a condition to the extent that a positive clinical benefit isobserved. Thus, a course of therapy includes prophylactic treatment, andfurther pertains generally to the therapy of a human subject. Forexample, the treatment of sperm oxidative stress and/or male infertilityassociated with sperm oxidative stress includes a reduction in the rateof production, rate of proliferation or the concentration of ROS insperm cells, which can result in a halt in the rate of infertility.Treatment includes combination treatments and therapies, in which two ormore treatments or therapies modalities are combined, for example,sequentially or simultaneously. Examples of treatments and therapiesinclude, but are not limited to, antioxidant combination therapy with orwithout the use of anti-inflammatory agents, surgery, employing ARTand/or other drug treatments generally known to improve rate ofpregnancy.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of an active compound, or a material, composition or dosageform comprising an antioxidant, which is effective for producing thedesired therapeutic effect, commensurate with a reasonable benefit/riskratio to avoid excess concentration of such drugs to the extent that itwould neutralize its expected therapeutic effects.

The term “vitamin C and/or its derivatives,” except as individuallyrecited, is meant to include ascorbic acid, a mineral ascorbate or amulti-mineral ascorbate, calcium ascorbate, magnesium ascorbate, zincascorbate, potassium ascorbate, sodium ascorbate, molybdenum ascorbate,chromium ascorbate, vitamin C Complex, Ascorbyl palmitate.

The term “vitamin E and/or its derivatives” is meant to include allvariations of vitamin E, including but not limited the eightantioxidants commonly known as vitamin E; four tocopherols (alpha-,beta-, gamma-, and delta-) and four tocotrienols (alpha-, beta-, gamma-,and delta-).

The term “zinc” is meant to include all variations of zinc including butnot limited to elemental zinc, zinc sulphate, zinc picolinate, zinccitrate, zinc acetate, zinc glycerate, and zinc monomethionine.

The invention provides a combination therapy for improving fertility inmammals particularly among those suffering from excess oxidative stressat their reproductive cell levels. At least one aspect of the presentinvention provides treating infertility among any mammal including butnot limited to land and aquatic animals such as primates, horses,donkeys, sheep, cats, dogs, pigs etc. In a more preferred embodiment,the mammal is a male human, horse or cow and in the most preferredembodiment the mammal is human.

In another aspect of the present invention the presently disclosedtreatment reduces male infertility associated with sperm oxidativestress. The invention provides a scientifically validated nutritionalblend for mammalian male to reduce sperm oxidative stress andinfertility associated with such condition and further improve the spermgenomic integrity. The present combination of vitamins, minerals andamino acids reduces overall fragmentation of sperm cells and furthersecure higher rate of pregnancy among mammalian couples whose malecounterpart suffers from infertility.

Those of ordinary skill in the art understand that sperm is highlysusceptible to free radical or oxidative damage from environmentaltoxicants and natural aging. In at least one embodiment of the presentinvention vitamins C and E, zinc, and selenium, all of which are potentantioxidants that help improve sperm counts and quality, are used toneutralize the formation of excess ROS and maintain normal DNA makeup.Zinc and B vitamins (B6, B12 and folate) are critical nutrients in malereproductive systems for hormone metabolism, sperm formation andmotility. However, synergistic combination of such ingredients forpurposes of treating male infertility secondary to SOS has not beenclinically proven. At least one aspect of the present invention,describes a profertility combination of such ingredients for treatingmale infertility associated with SOS.

In another aspect, the profertility combination of the present inventionemploys a source of carnitine. Such source includes L-carnitine,acetyl-L-carnitine or priopionyl-L-carnitine and all pharmaceuticallyacceptable salts thereof. The preferred embodiment of this aspect of theinvention employs priopionyl-L-carnitine, hydrochloride, fummurate ortarterate salts thereof.

Another aspect of the present invention provides for a component thatwould have a synergistic action of the combinations. The distinctcombinations are useful for couples who have failed to produce a healthyfertilized oocyte during at least a period ranging from three (3) to six(6) months prior to any fertilization attempts. Such couples generallyinclude male subjects exposed to occupational toxic agents such asstyrene, as well as those who may be subject to environmental, chemical,radiation, and heat exposure.

In one embodiment, the male human subject of the present invention isselected from the group consisting of a subject with increased levels ofsperm DNA oxidation, reduced fertility of unknown origin; a subjecthaving undergone vasectomy reversal; a subject with a reproductive tractinfection such as epididymitis; and a subject having a varicocele.

Previous human studies assessing male subjects with increased levels ofmalondialdehyde or other biochemical markers of oxidative stress; asmoker; a subject with reduced fertility, including reduced fertilitydue to poor sperm motility have all failed to substantiate the use ofother antioxidants' combinations in treating DNA fragmentation secondaryto sperm oxidative stress. See Tremellen supra.

The present inventor has unexpectedly discovered that the instantlyclaimed blend of antioxidants drastically reduce sperm DNA fragmentationfrom the pretreatment baseline and increase rate of pregnancy.

Methods are known in the art for assessing the extent of free radicaldamage to sperm. For example, the thiobarbituric acid reactivesubstances (“TBARs”) assay (which involves the measurement ofmalondialdehyde, a marker of sperm membrane oxidation) or LPO-856spectrophotometric assay may be used. These methods are described forexample in Gomez et al International Journal of Andrology 21(2), 81-96(1998). Other methods include measurement of DNA Fragmentation Index(“DFI”) using SCSA, SCD test, DBD-FISH test, and 8-OH-dG Assay.

In one embodiment, the administration of the present combination therapyto the subject results in a reduction in free radical damage to sperm'sDNA of at least 2.5%, 5%, 8%, 10%, 12.5%, 15%, preferably 20% and mostpreferably 30%, when measured by a suitable sperm parameter analysis,preferably evaluated or expressed as DFI. In at least anotherembodiment, the administration of the present combination therapy to themale subjects results in a significant improvement in the degree of DNAdamage secondary to SOS, when compared to the subject's own pretreatmentvalues, by at least 1, 2.5%, 5%, 8%, 10%, 12.5%, 15%, 20%, 30%, andpreferably 50%, wherein such improvement advances the pregnancy of afemale partner. In a preferred embodiment, the administration of theantioxidant composition to a male subject results in a reduction inpretreatment baseline levels of the subject's DFI measurement by atleast an absolute 2.5% (i.e. the actual difference in DFI measurementbefore and after the combination therapy).

The anti-oxidant agent in the various embodiments of the presentinvention may be one or more individual antioxidant(s). In this regard,an antioxidant is a molecule that can directly or indirectly reduce thedamaging effects of oxygen and/or free-radicals in cells, and includesmolecules that react with oxygen, or molecules that may protect against,and/or react with, a free radical.

In one embodiment, the anti-oxidant agent is selected from one or moreof the group consisting of a vitamin C; vitamin E; β-carotenoid,including lycopene (a carotenoid derived from the tomato), lutein; afolic acid or derivative, selenium; zinc; L-carnitine, a prodrug thereofsuch as propionyl-L-carnitine; acety-L-carnitine; N-acetylcysteine;glutathione; gluthathionine; pyruvate; Coenzyme Q10, astaxanthin andhypotaurine; alpha-lipoic acid or a salt (if applicable), or apharmaceutically acceptable derivative of any of the aforementionedagents. Other anti-oxidants are generally as described in Agarwal et al,Role of Antioxidants in Treatment of Male Infertility: an Overview ofthe Literature, Reproductive Biomedicine Online, 8(6), 616-62 (2004).Compounds such as gluthathione, astaxanthin or Coenzyme Q10 may beadministered parenterally to enhance the ultimate clinicalantioxidation.

The effective amount of the one or more anti-oxidant agents in thevarious embodiments of the present invention is not particularlylimited, so long as it has the desired or therapeutic effect, and willdepend upon the particular anti-oxidant(s) administered. In a preferredembodiment, the present invention contains tablets having vitamin E(d-alpha-tocopheryl acetate) in ranges of 50 to 1500 I.U., with a usualrange of 200 to 1200 I.U., and typically 200 to 800 I.U.; vitamin C(ascorbic acid, or a salt thereof) in ranges of 10 to 1000 mg, with ausual range of 50 to 500 mg and typically 90 to 200 or 400 mg; seleniumin ranges of 10 to 250 μg; zinc in amounts of about 5 to 100 mg; folicacid in amounts of about 0.1-1 mg; lycopene in amounts of about 0.5 to20 mg, with usual ranges of 1 to 10 mg, a carnitine source in amounts ofabout 1 to 5 grams, with a usual range of 2 to 3 grams; and optionallyglutathione in amounts of 100 to 1000 mg, with a usual range of 400 to600 mg, preferably administered parenterally. In another aspect of thisinvention, the combination is supplemented with alpha-lipoic acid inamounts of about 50 to 600 mg.

In another aspect of the present invention, the combination therapy canoptionally be supplemented with an aromatase inhibitor. Preferredaromatase inhibitor agents include natural and synthetic compounds thatare able to modulate and reduce the activity of Aromatase, an enzymefound in the liver, which is responsible for the conversion of theandrogens such as androstendione and testosterone into the estrogenssuch as estrone and estradiol. By inhibiting aromatase the body producesless estrogen and maintains a higher testosterone state. Suitablearomatase inhibitors include natural flavanoids such as chrysin,apigenin, indole-3-carbinols or synthetic compounds including but notlimited to aminoglutethimide, anastrozole or6-methylenandrosta-1,4-diene-3,17-dione.

In another embodiment, the anti-oxidant agent administered to thesubject is a combination of the following anti-oxidant agents: vitaminC, vitamin E, selenium, zinc, and propionyl-L-carnitine. In yet anotherembodiment, the combination of the anti-oxidant agents consistsessentially of vitamin C, vitamin E, selenium, zinc,propionyl-L-carnitine and folic acid. In yet another embodiment, thecombination of the anti-oxidant agents consists of vitamin C, vitamin E,selenium, zinc, propionyl-L-carnitine and lycopene. In yet anotherembodiment, the combination of the anti-oxidant agents consistsessentially of vitamin C, vitamin E, selenium, zinc, L-carnitine, folicacid and lycopene. In yet another embodiment, the combination of theanti-oxidant agents consists of vitamin C, vitamin E, selenium, zinc,acetyl-L-carnitine, folic acid and lycopene, and suitable excipients. Inthe most preferred embodiment, the combination of the anti-oxidantagents consists of vitamin C, vitamin E, selenium, zinc,acetyl-L-carnitine, proionyl-L-carnitine, folate or folic acid andlycopene and suitable excipients.

A suitable formulation (referred to as the Fertilix™ formulation)contains vitamin E (d-alpha-tocopheryl acetate) vitamin C (ascorbic acidor a salt thereof), selenium, zinc, folate or folic acid, lycopene,propionyl-L-carnitine, and acetyl-L-carnitine.

At least one embodiment of such formulation essentially contains vitaminE (d-alpha-tocopheryl acetate) 200 I.U.; vitamin C (ascorbic acid or asalt thereof) 90-200 mg, selenium 55 μg, zinc 15 mg, folate or folicacid 0.5 mg, lycopene 5 mg, propionyl-L-carnitine 500 mg, andacetyl-L-carnitine 1 gram. Another embodiment contains vitamin C(ascorbic acid or a salt thereof) in amount of 90 mg. Yet in anotherembodiment, the formulation further contains lipoic acid in amount of 60mg.

In one embodiment, the duration for the treatment is at least for six(6) weeks to about twelve (12) months. In another embodiment, theduration of the therapy is for three (3) months prior any fertilizationattempts. In further embodiments, the duration of the treatment regimeis for six (6) months prior any fertilization attempts. In this regard,a suitable therapy for either of the following formulations is twoadministrations per day of one of the combinations, formulations, orcompositions described above for a period of at least three (3) monthsprior to any fertilization attempts. However, it will be appreciatedthat the administration of the disclosed formulation and the otheragents in the various embodiments of the present invention may be withinany time and frequency suitable to produce the desired effect.

The anti-oxidant and the other agents of the present invention includethose suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal and/or parenteral administration.Regardless of the route of administration selected, the combination ofthe ingredients are formulated into pharmaceutically-acceptable dosageforms by conventional methods known to those of skill in the art.

The amount of the active antioxidants which will be combined with acarrier material to produce a single dosage form will generally be thatamount of the active ingredient(s) which is the optimal dose effectiveto produce the therapeutic effect, herein as measured by DFI.

Methods of preparing pharmaceutical formulations or compositions includethe step of bringing the antioxidants and/or other suitable activeingredients into association with the carrier and, optionally, one ormore accessory ingredients. In general, the formulations are prepared byuniformly and intimately bringing the active ingredient(s) intoassociation with liquid carriers, or finely divided solid carriers, orboth, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, microparticles, nanoparticles or as a solution or a suspensionin an aqueous or nonaqueous liquid, or as an oil-in-water orwater-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles(using an inert base, such as gelatin and glycerin, or sucrose andacacia) and/or as mouth washes and the like, each containing apredetermined amount of the active ingredient(s).

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, powders, granules and the like), theprodrug(s), antioxidant(s) and/or other suitable pharmaceutically activeingredient(s) (in their micronized, microparticle or nanoparticle forms)is/are mixed with one or more pharmaceutically-acceptable carriers, suchas cellulose, magnesium stearate, silicon dioxide, sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol,cellulose and/or silicic acid; (2) binders, such as, for example,carboxymethyl-cellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, silicon dioxide, other types of silicates,and sodium carbonate; (5) solution retarding agents, such as paraffin;(6) absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents.

In the case of capsules, tablets and pills, the pharmaceuticalcompositions may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as cellulose, magnesium stearate,silicon dioxide, lactose or milk sugars, with or without high molecularweight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered activeingredient(s) moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow, extended or controlled release of the desiredantioxident(s) therein using, for example, hydroxypropylmethyl cellulosein varying proportions to provide the desired release profile, otherpolymer matrices, nanoparticles and/or microspheres. They may besterilized by for example, filtration through a bacteria-retainingfilter.

Liquid dosage forms for oral administration of the active ingredient(s)include pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient(s), the liquid dosage forms may contain inert diluentscommonly used in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethylacetate, butyl alcohol, benzyl benzoate, propylene glycol,glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, amyl alcohol, tetrahydrofurylpolyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents. Suspensions, inaddition to the active ingredient(s), may contain suspending agents as,for example, ethoxylated alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, bentonite, agar and tragacanth, andmixtures thereof.

Dosage forms for the topical or transdermal administration of the activeingredient(s) include powders sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The activeingredient(s) may be mixed under sterile conditions withpharmaceutically-acceptable carrier, and with any buffers, orpropellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to theactive ingredient(s), excipients, such as animal and vegetable fats,oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof. Powders and sprays can contain, in additionto the active ingredient(s), excipients such as lactose, talc, silicicacid, aluminum hydroxide, calcium silicates and polyamide powder, ormixtures of these substances. Sprays and inhalers can additionallycontain customary propellants such as chlorofluorohydrocarbons andvolatile unsubstituted hydrocarbons, such as butane and propane.

Compositions of the present invention may be administered in intranasalform via topical use of suitable intranasal vehicles, or via transdermalroutes, using those forms of transdermal skin patches well known tothose of ordinary skill in the art. A transdermal delivery systemprovides for continuous administration throughout the treatment regimen.Transdermal patches have the added advantage of providing controlleddelivery of the active ingredient(s) to the body. Such dosage forms canbe made by dissolving, dispersing or otherwise incorporating the activeingredient(s) in a proper medium, such as an elastomeric matrixmaterial. Absorption enhancers can also be used to increase the flux ofthe active ingredient(s) across the skin. The rate of such flux can becontrolled by either providing a rate-controlling membrane or dispersingthe active ingredient(s) in a polymer matrix or gel.

Another mode of delivery for present combination of the presentinvention may be delivery via the use of targeting compounds.Accordingly a nanoparticulate or microparticulate blend of theantioxidant(s) in proper ratios may be coupled with soluble polymers astargetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamide-phenol,polyhydroxy-ethylaspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues, polylactic acid, polyglycolic acid,copolymers of polyactic and polyglycolic acid, polyepsilon caprolactone,polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacrylates and crosslinked or amphipathicblock copolymers of hydrogels.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise the antioxidant(s) in combination with one ormore pharmaceutically-acceptable sterile isotonic aqueous or nonaqueoussolutions, suspensions or emulsions, or sterile powders which may bereconstituted into sterile injectable solutions or dispersions justprior to use, which may contain the combination of the antioxidants,minerals and vitamins, buffers, solutes which render the formulationisotonic with the blood of the intended recipient or suspending orthickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, dextrose, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof.Proper fluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize, and by the use of surfactants.

These compositions may also contain adjuvants such as wetting agents,emulsifying agents and dispersing agents. It may also be desirable toinclude isotonic agents, such as sugars, sodium chloride, and the likein the compositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monostearate and gelatin.

Injectable depot forms are made by forming microencapsule matrices ofthe antioxidant(s), minerals and vitamins in biodegradable polymers suchas polylactide-polyglycolide. Depending on the ratio of the activeingredient(s) to polymer, and the nature of the particular polymeremployed, the rate of release of the active ingredient(s) can becontrolled. Examples of other biodegradable polymers includepoly(orthoesters) and poly(anhydrides). The injectable materials can besterilized for example, by filtration through a bacterial-retainingfilter.

The pharmaceutical compositions of the present invention may also beused in the form of veterinary formulations for treating infertilecattle, herd, or other infertile mammals, including those adapted forthe following: (1) oral administration, for example, drenches (aqueousor nonaqueous solutions or suspensions), tablets, boluses, powders,granules or pellets for admixture with feed stuffs, pastes forapplication to the tongue; (2) parenteral administration, for “ampule,”by subcutaneous, intramuscular or intravenous injection as, for example,a sterile solution or suspension or, when appropriate, by injectionwhere a suspension or solution is introduced to the animal; (3) topicalapplication, for example, as a cream, ointment or spray applied to theskin; or any other methods fit to by those of ordinary skill in the artfor administration to a region of interest.

Formulation of the agents of the present invention and theiradministration may be facilitated by an easy to use kit to ameliorateand assist patients to follow the treatment regimen. As discussedpreviously herein, the agents in the various embodiments of the presentinvention may be administered jointly or separately in the form of oralpreparations (for example solid preparations such as tablets, capsules,granules or powders; liquid preparations such as syrup, emulsions orsuspensions).

Compositions containing the anti-oxidant agent and the other agentsseparately or jointly in the various embodiments of the presentinvention may also contain a preservative, stabilizer, dispersing agent,pH controller or isotonic agent Examples of suitable preservatives inthe various embodiments of the present invention are glycerin, propyleneglycol, phenol or benzyl alcohol. Examples of suitable stabilizers inthe various embodiments of the present invention are dextran, gelatin,or alpha-thioglycerin.

Examples of suitable dispersing agents in the various embodiments of thepresent invention include polyoxyethylene (20), sorbitan mono-oleate(Tween 80), sorbitan sesquioleate (Span 30), polyoxyethylene (160)polyoxypropylene (30) glycol (Pluronic F68) or polyoxyethylenehydrogenated castor oil 60. Examples of suitable pH controllers in thevarious embodiments of the present invention include hydrochloric acid,sodium hydroxide and the like. Examples of suitable isotonic agents areglucose, D-sorbitol or D-mannitol.

As discussed previously herein, the present invention is also suitablefor reducing the generation of ROS in the male reproductive tract and/orsemen. In this regard, the male reproductive tract will be understood toinclude the epididymis, the penis, the prostate gland, the seminalvesicles, the testes, the vas deferens and semen. Methods fordetermining the level of free radicals in the male reproductive tractare known in the art. For example, free radical production by sperm orseminal leukocytes can be measured directly using chemiluminescenceassays known in the art. Alternatively, assays that measure free radicalrelated damage to sperm lipid membrane may be used. For example, theTBARs assay (which involves the measurement of malondialdehyde, a markerof sperm membrane oxidation) or LPO-586 spectrophotometric assay may beused. These methods are described in Gomez et al (1998) InternationalJournal of Andrology 21(2):81-96.

In one embodiment, the level of male fertility track is assessed by theway of suitable inflammatory indices. For example, one of ordinary skillin the art would with to assess the presence or absence of inflammatorycytokine, like one or more of IL-I, IL-6, IL-, TNF-α and Interferon-γ.Methods for determining the level of an inflammatory cytokine in themale reproductive tract are known in the art, such ELISA assays fordetection of pro-inflammatory cytokines such as IL-I, IL-6, IL-8, TNF-αand Interferon-γ, as described in Depuydt et al. J Andrology 17(61),699-707 (1996) or Maegawa et al J Reprod Immunology, 54, 33-42 (2002),and Nallella et al. Urology 64(5), 1010-3 (2004).

Those of ordinary skill in the art can also employ other methods ofassessing the “sperm function.” The term as used herein includes any keycomponent of sperm physiology and includes swimming activity towards theoocyte (motility), ability to undergo capacitation to penetrate theoocyte's outer coat (zona pellucida) and fuse with the oocyte membrane,and maintenance of sperm DNA integrity to form a functional malepro-nucleus at syngamy. Methods for determining the level of spermfunction are known in the art. For example, suitable methods aredescribed in detail within the World Health Organization (WHO)laboratory manual for the examination of human semen and sperm-cervicalmucous interaction. 4th edition. Cambridge University Press 1999.

In at least one embodiment of the present invention, the sperm DNAfragmentation is assessed by SCSA. Evenson D P, et al, RelationshipBetween Assisted Reproductive Techniques (ART) outcome and Status ofChromatin Integrity as Measured by the Sperm Chromatin Structure Assay(SCSA). Hum. Reprod., 15, 1717-1722 (2000). At least one indicator ofthe clinical benefits of the claimed methodology is marked improvementof the DFI.

In principle, spermatozoa chromatin is different in nature from somaticcells chromatin. This difference is due to ploidy and DNA packaging,which is influenced by the replacement of histones in spermatocytes bytransition proteins and then substitution of histone by protamine inspermatides. Green et al, Synthesis and Processing of MammalianProtamines and Transition Proteins. Mol. Repr Dev 37, 255-263 (1994).Histones are the chief protein components of chromatin that coils aroundDNA enabling it to fit the large genomic information inside cell nuclei.Without histones, the unwound DNA in chromosomes would be very long.Replacement of histones by protamine are generally known to contributeto sperm's head condensation and DNA stabilization. See Id. Those ofordinary skill in the art can appreciate that DNA fragmentation levelsabove 30% as measured by SCSA are not compatible with initiation andmaintenance of a term pregnancy. See Larson supra. SCSA test values,expressed as DFI, are significantly correlated with pregnancy rate invivo and in vitro. In studies that included more than 25 couplesundergoing in vitro fertilization and intracytoplasmic sperm injectioncycles, no term pregnancy occurred when the DFI measurement was morethan 27% in the semen samples utilized in these cycles. See Larsonsupra.

In the preferred embodiment of the present invention, baseline DFI ismeasured prior to the administration of the antioxidant regimen toassess a fertility capacity of male subjects. In the present invention,DFI values in combination with other subjective or objective signs ofinfertility would be used as the criteria to determine couplespredisposition to failed pregnancy. Once study couples have undergonethe baseline evaluation, they will receive a three (3) months course oftherapy of the claimed antioxidant formulation in a kit containing othereducational material regarding their therapy.

The effectiveness of the treatment course is then substantiated bystatistically significant reduction in DFI and further the actualpregnancy of the couple's female subject. Those of ordinary skill in theart can appreciate measurements of other sperm parameters during theperiod of the trial to assess treatment efficacy. Such parameters caninclude sperm count, sperm density, motility, membrane integrity,testosterone or other hormonal levels if so desired, and other DNAdamage techniques at various intervals including the entry period, 6weeks, and/or 12 week time points. However, the actual clinical endpoint can be evaluated in view of other indicators including but notlimited to fertilization rates among treatment group, embryo orblastocyst quality, number of viable embryos or blastocyst for transfer,number of miscarriages and the occurrence rate thereof.

An effective amount of the antioxidant agents and the other agents maybe appropriately chosen, depending upon, for example, the type andextent of reduced fertility to be treated, the age and body weight ofthe subject, the DFI measurement, the frequency of administration, andthe presence of other active agents. The instant antioxidant, mineraland vitamin combination, and/or the anti-aromatase agent may beadministered to the subject separately or in combination via separateroutes of administrations.

Accordingly, in another embodiment the present invention provides acombination product for improving sperm function in a male subject, thecombination kit product including the following components: (a) acomposition containing the present antioxidant, mineral and vitamincombination; and/or (b) an aromatase inhibitor agent, and/or (c) anotheradjuvant agent such as gluthothione or Coenzyme Q10 with (d) educationalinformation and direction of use.

In yet another embodiment, the inventor describes the use of a carnitinesource in the combinations described herein. L-carnitine,acetyl-L-carnitine and propionyl-L-carnitine are endogenous ligands thatplay essential role in transporting fatty acids into the mitochondria,where they are oxidized to produce energy. The carnitine pool isparticularly important to spermatozoa as they are rich in mitochondriagenerating energy especially to support their motility. Carnitines arealso scavengers of oxygen free radicals in mammalian tissues. Thus, thebiochemical and antioxidant role of carnitines make them vitallyimportant molecules in the overall health of the spermatozoa.

Propionyl-L-carnitine is known in the art to increase cellular levels ofL-carnitine by binding to the enzyme carnitine acetyltransferase (CAT)which is subsequently converted into propionyl-coenzyme A and freecarnitine. Propionyl-L-carnitine is also rapidly absorbed after oraladministration reaching peak concentrations in about 1 hour followed bya rise in L-carnitine plasma levels 2-6 hours after the administration.

WO publication 03/084526 have previously shown the impact of suchprodrugs in sperm motility and concentration. However, theacetyl-L-carnitine and propyl-L-carnitine alone or in combination havenot been described in male prenatal compositions have not beendescribed. In at least one aspect of the present invention, theinventors believe that the exogenous propionyl-L-carnitinesupplementation alone will more effectively address any carnitinedeficiency in infertile men.

The forgoing examples are used to further describe the inventionemploying at least one embodiment of the presently disclosedcombination, the Fertilix™ profertility therapy Those of ordinary skillin the art can appreciate that patients who had received IVF before andduring, as well as, those patients resorting to other fertilizationattempts, would benefit from the presently disclosed treatment regimenby improving their embryonic quality and reducing the complicationsduring the pregnancy term. At least one embodiment of the presentinvention focus the treatment regimen on the male partners. Yet anotherembodiment of the invention, include independently or simultaneouslyadministering the regimen to the female partners.

With well over 3 million men currently experiencing male relatedinfertility in the United States. Traditionally male infertilitytreatment has not endeavored to ameliorate the underlying cause ofinfertility but rather used “mechanical” techniques such asintra-uterine insemination or IVF-ICSI to bypass the defect in spermfunction. While these two techniques are undeniably successful in alarge proportion of patients, they simply do not work or have verylimited efficiency in other couples. It is likely that in many cases,sperm DNA fragmentation is responsible for the poor pregnancy outcomedespite ART treatment. The present invention provides treatments thatcan prophylactically treat sperm DNA fragmentation and is likely toboost both natural and ART related pregnancy rates.

A total of two (2) separate human trials are envisioned for furtherassessing the efficacy of the combination therapy instantly disclosed.Prior experiences with antioxidant combinations have not provided anyevidence that antioxidant combinations can reduce sperm's DNAfragmentation secondary to oxidative stress. See Tremelton supra.Contrary to such scientific body of evidence, the present treatmentregimen unexpectedly show a significant reduction of DFI after three (3)months of therapy.

In assessing the efficacy of other prior art antioxidant combinations,one of ordinary skill in the art would appreciate that even though therate of pregnancy outcomes might have improved in the antioxidanttreatment groups; the measured sperm parameters for the treatment groupwas not affected and the treatment had no effect on sperm concentration,motility or morphology. See Tremelton supra at pgs 219-220. Furthermore,the LPO-586 assay for lipid peroxidation damage did not detect anysignificant difference in levels of free radical damage to the spermmembrane of the subjects. See Id. In fact, prior art formulations failedto show a correlation between SOS or sperm DNA damage and pregnancyrates since the sperm DNA damage assays employed failed to show adifference between the treatment and the placebo groups. See Id.

The present invention provides a new combination of antioxidants with anunexpected reduction in sperm's DNA fragmentation due to local oxidativestress. Unexpected results extend to the inventors observation ofimproved quality of the sperm parameters, which when coupled withsuccessful pregnancies, confirms the efficacy of Fertilix™ therapy intreating male infertility associated with SOS.

Example 1

First combination for men is prepared according to the followingformula:

TABLE I d-alpha-tocopheryl acetate 200 IU Ascorbic acid 200 mg Selenium55 μg Zinc 15 mg Folic acid 0.5 mg Lycopene 5 mg Propionyl-L-Carnitine0.5 g Excipients As needed

Example 2

A second combination is prepared according to the following formula:

TABLE 2 Vitamin E 200 IU Vitamin C 200 mg Selenium 55 μg Zinc 15 mgFolic acid 0.5 mg Lycopene 5 mg Acetyl-L-Carnitine 1.0 gPropionyl-L-Carnitine 0.5 g Excipients As needed

Example 3

Another formulation is prepared according to the following formula:

TABLE 3 Vitamin E 200 IU Vitamin C 200 mg Selenium 55 μg Zinc 15 mgFolic acid 0.5 mg Lycopene 5 mg L-Carnitine 1 g Excipients As needed

Example 4

Another formulation is prepared according to the following formula:

TABLE 4 Vitamin E 200 IU Vitamin C 200 mg Selenium 55 μg Zinc 15 mgFolic acid 0.5 mg Lycopene 5 mg L-Carnitine 1.0 g Acetyl-L-Carnitine 0.5g Excipients As needed

Example 5

Another formulation is prepared according to the following formula:

TABLE 5 Vitamin E 200 IU Vitamin C 200 mg Selenium 55 μg Zinc 15 mgFolic acid 0.5 mg Lycopene 5 mg L-Carnitine 1.0 g Propionyl-L-Carnitine0.5 g Excipients As needed

Example 6

Another formulation for men is prepared according to the followingformula:

TABLE 6 Vitamin E 200 IU Ascorbic acid 90 mg Selenium 55 μg Zinc 15 mgFolic acid 0.5 mg Lycopene 5 mg Propionyl-L-Carnitine 0.5 g ExcipientsAs needed

Example 7

Yet, another formulation is prepared according to the following formula:

TABLE 7 Vitamin E 200 IU Vitamin C 90 mg Selenium 55 μg Zinc 15 mg Folicacid 0.5 mg Lycopene 5 mg Acetyl-L-Carnitine 0.75 gPropionyl-L-Carnitine 0.5 g Excipients As needed

Example 8

Another formulation is prepared according to the following formula:

TABLE 8 Vitamin E 200 IU Vitamin C 90 mg Selenium 55 μg Zinc 15 mg Folicacid 0.5 mg Lycopene 5 mg L-Carnitine 1 g Excipients As needed

Example 9

Another formulation is prepared according to the following formula:

TABLE 9 Vitamin E 200 IU Vitamin C 90 mg Selenium 55 μg Zinc 15 mg Folicacid 0.5 mg Lycopene 5 mg L-Carnitine 1.0 g Acetyl-L-Carnitine 0.5 gExcipients As needed

Example 10

Another formulation is prepared according to the following formula:

TABLE 10 Vitamin E 200 IU Vitamin C 90 mg Selenium 55 μg Zinc 15 mgFolic acid 0.5 mg Lycopene 5 mg L-Carnitine 1.0 g Propionyl-L-Carnitine0.5 g Excipients As needed

Example 11

Couple DP underwent multiple cycles of IVF that resulted in no viablepregnancy. The sperm DNA fragmentation index of the male subject wasmeasured by SCSA to be 27%. The male partner then started on a twice aday combination therapy described in Example 3, Table 3 for a period ofthree (3) months. The combination contained vitamin E(d-alpha-tocopheryl acetate) 200 I.U., vitamin C 200 mg, selenium 55 μg,zinc 15 mg, folic acid 0.1 mg, L-carnitine 1 g, and lycopene 5 mg.

The post treatment DFI measurement showed an absolute 7.4% reduction inthe DFI levels amounting to a 27.4% improvement from the pretreatmentbaseline DFI levels. Upon completion of the therapeutic regimen, eventhough no in vitro embryo assessment was done, the female partner becamepregnant and gave birth to a healthy child nine (9) months following thetermination of the anti-oxidant therapy.

Example 12

In another trial, the same couple used the same treatment regimen asdescribed in Example 2. The male's sperm DFI measurement indicated afree radical damage of 39.6% prior to the initiation of the antioxidantcombination therapy. The DFI measurement at least three (3) months afterthe initial treatment was measured as 27.2 showing an unexpectedimprovement of at least an absolute 12.4% reduction in DNA fragmentationindex amounting to a 31% improvement from the pretreatment baseline DFIlevels. While no in vitro embryo assessment was done, the female partnerbecame pregnant and gave birth to a healthy child nine (9) monthsthereafter.

While the invention has been described with references to specificembodiments, modifications and variations of the invention may beconstrued without departing from the scope of the invention, which isdefined in the following claims.

1. A method of treating male infertility secondary to sperm oxidativestress in a patient suffering from sperm DNA fragmentation Indexof >30%, comprising administering to said patient a composition for aperiod of up to three months wherein said composition comprising,vitamin E; vitamin C; selenium; zinc; lycopene or lipoic acid; at leastone source of carnitine, a folate or folic acid or suitable saltsthereof and a pharmaceutically suitable carrier or excipient, andwherein the composition is substantially free of any traces of vitaminA, vitamin K, and garlic, and further wherein the DNA fragmentationIndex is reduced by at least 2.5% after the three month time period. 2.The method of claim 1, wherein said folic acid is administered in anamount ranging from about 0.1 mg to about 5.0 mg.
 3. The method of claim1, wherein the vitamin C source is ascorbic acid and vitamin E isselected from the group consisting of a tocopherol or a tocotrienol or acombination thereof.
 4. The method of claim 1, wherein the vitamin Csource is a vitamin C derivative selected from the group consisting ofcalcium ascorbate, magnesium ascorbate, zinc ascorbate, potassiumascorbate, sodium ascorbate, a mineral ascorbate or a multi-mineralascorbate and combinations thereof.
 5. The method of claim 3, whereinthe vitamin C is administered to the patient in an amount ranging fromabout 10 mg to about 1000 mg.
 6. The method of claim 4, wherein thecomposition is in a tablet dosage form selected from the groupconsisting of an immediate release tablet, an extended or pulsatilerelease tablet, a chewable tablet, a chewable lozenge, a quick chew, aquick dissolve and combinations thereof.
 7. A method of prophylacticallytreating a disease associated with increased levels of reactiveoxidative species in sperm comprising administering to a male subject inneed thereof, a composition comprising an effective amount of vitamin E;vitamin C; selenium; zinc; lycopene or lipoic acid; at least one sourceof carnitine, a folate or folic acid or suitable salts thereof, as thesole active ingredients and a pharmaceutically suitable carrier orexcipient wherein the composition is substantially free of vitamin A,vitamin K, garlic.
 8. A method of prophylactically treating formation ofan nonviable zygote comprising administering to a male subject in needthereof, a composition comprising an effective amount of vitamin E;vitamin C; selenium; zinc; lycopene or lipoic acid; at least one sourceof carnitine, a folate or folic acid or suitable salts thereof, as thesole active ingredients and a pharmaceutically suitable carrier orexcipient wherein the composition is substantially free of vitamin A,vitamin K, garlic.
 9. The composition consisting of the following activeingredients: (a) from 200 I.U. to 600 I.U. of vitamin E selected fromthe group consisting of alpha-tocopherol, beta-tocopherol,gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol,gamma-tocopherol, delta-tocopherol, and any combinations thereof; (b)from 75 mg to 100 mg vitamin C; (c) from 45 μg to 60 μg selenium; (d)from 5 mg to 15 mg zinc in suitable salt form; (e) from 0.25 mg to 0.60mg of folic acid; (f) from 5 mg to 20 mg lycopene; (g) from 100 mg to800 mg propionyl-L-carnitine; and (h) from 100 mg to 800 mgacetyl-L-carnitine, and at least one pharmaceutically suitable carrieror excipient.
 10. A composition consisting of the active ingredients:(a) from about 50 I.U. to about 1000 I.U. of vitamin E selected from thegroup consisting of alpha-tocopherol, beta-tocopherol, gamma-tocopherol,delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocopherol,delta-tocopherol, and any combinations thereof; (b) from about 50 mg toabout 400 mg vitamin C; (c) from about 45 μg to about 65 μg selenium;(d) from about 10 mg to about 30 mg zinc in its suitable salt form, (e)from about 0.25 mg to about 0.75 mg of folic acid; (f) from about 2.5 mgto about 20 mg lycopene; (g) from about 100 mg to about 2000 mg of acarnitine source selected from the group consisting ofpropionyl-L-carnitine; acetyl-L-carnitine; or a combination thereof; andat least one pharmaceutically suitable carrier or excipient.
 11. Acomposition consisting of the active ingredients: (a) from about 50 I.U.to about 1000 I.U. of vitamin E selected from the group consisting ofalpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol,alpha-tocotrienol, beta-tocotrienol, gamma-tocopherol, delta-tocopherol,and any combinations thereof (b) from about 50 mg to about 400 mgvitamin C; (c) from about 45 μg to about 65 μg selenium: (d) from about10 mg to about 30 mg zinc in its suitable salt form, (e) from about 0.25mg to about 0.75 mg of folic acid; (f) from about 2.5 mg to about 20 mglycopene; (g) from about 100 mg to about 2000 mg of a carnitine sourceselected from the group consisting of propionyl-L-carnitine;acetyl-L-carnitine; or a combination thereof; (h) N-acetyl-cysteine andat least one pharmaceutically suitable carrier or excipient.